When penicillin became widely available during the second world war, it was a medical miracle, rapidly vanquishing the biggest wartime killer-infected wounds. Discovered initially by a French medical student, Ernest Duchesne, in 1896, and then rediscovered by Scottish physician Alexander Fleming in 1928, the product of the soil hold Penicillium crippled many types of disease-causing bacteria. But just four years after drug companies began mass-producing penicillin in 1943, microbes began appearing that could resist it.
The first bug to battle penicillin was Staphylococcus aureus. This bacterium is often a harmless passenger in the human body, but it can cause illness, such as pneumonia or toxic shock syndrome, when it overgrows or produces a toxin. In 1967, another type of penicillin-resistant pneumonia, caused by Streptococcus pneumoniae and called pneumococcus, surfaced in a remote village in Papua New Guinea. At about the same time, American military personnel in Southeast Asia were acquiring penicillin-resistant gonorrhea from prostitutes. By 1976, when the soldiers had come home, they brought the new strain of gonorrhea with them, and physicians had to find new drugs to treat it. In 1983, a hospital-acquired intestinal infection caused by the bacterium Enterococcus faecium joined the list of bugs that outwit penicillin. Antibiotic resistance spreads fast. Between 1979 and 1987, for example, only 0.02 percent of pneumococcus strains infecting a large number of patients surveyed by the National Centers for Disease Control and Prevention (CDC) were penicillin-resistant. The CDC's survey included 13 hospitals in 12 states. By 1994, 6.6 percent of pneumococcus strains were resistant, according to a report in the Jun. 15, 1994, Journal of the American Medical Association by Robert F. Breiman, M.D., and colleagues at CDC. The agency also reports that in 1992, 13,300 hospital patients died of bacterial infections that were resistant to antibiotic treatment. (R. Lewis “The Rise of Antibiotic Resistant Infections” www.fda.gov). According to experts in the field such as Michael Blum, M.D. (medical officer in the Food and Drug Administration's division of anti-infective drug products), one of the main contributors of the alarming increase in antibiotic-resistant infections was a result of complacency: “There was complacency in the 1980s. The perception was that we had licked the bacterial infection problem. Drug companies weren't working on new agents. They were concentrating on other areas, such as viral infections. In the meantime, resistance increased to a number of commonly used antibiotics, possibly related to overuse of antibiotics. In the 1990s, we've come to a point for certain infections that we don't have agents available.” According to a report in the Apr. 28, 1994, New England Journal of Medicine, researchers have identified bacteria in patient samples that resist all currently available antibiotic drugs.
The increased prevalence of antibiotic resistance is an outcome of evolution. Any population of organisms, bacteria included, naturally includes variants with unusual traits, in this case the ability to withstand an antibiotic's attack on a microbe. When a person takes an antibiotic, the drug kills the defenseless bacteria, leaving behind—or “selecting,” in biological terms—those that can resist it. These renegade bacteria then multiply, increasing their numbers a million-fold in a day, becoming the predominant microorganism.
The antibiotic does not technically cause the resistance, but allows it to happen by creating a situation where an already existing variant can flourish. “Whenever antibiotics are used, there is selective pressure for resistance to occur. It builds upon itself. More and more organisms develop resistance to more and more drugs,” says Joe Cranston, Ph.D., Director of the Department of Drug Policy and Standards at the American Medical Association in Chicago. A patient can develop a drug-resistant infection either by contracting a resistant bug to begin with, or by having a resistant microbe emerge in the body once antibiotic treatment begins. Drug-resistant infections increase risk of death, and are often associated with prolonged hospital stays, and sometimes complications. These might necessitate removing part of a ravaged lung, or replacing a damaged heart valve.
Disease-causing microbes thwart antibiotics by interfering with their mechanism of action. For example, penicillin kills bacteria by attaching to their cell walls, then destroying a key part of the wall. The wall falls apart, and the bacterium dies. Resistant microbes, however, either alter their cell walls so penicillin can't bind or produce enzymes that dismantle the antibiotic. In another scenario, erythromycin attacks ribosomes, structures within a cell that enable it to make proteins. Resistant bacteria have slightly altered ribosomes to which the drug cannot bind. The ribosomal route is also how bacteria become resistant to the antibiotics tetracycline, streptomycin and gentamicin.
Mycobacterial Disease
Mycobacterial infections often manifest as diseases such as tuberculosis. Human infections caused by Mycobacteria have been widespread since ancient times, and tuberculosis remains a leading cause of death today. Although the incidence of the disease declined, in parallel with advancing standards of living, since the mid-nineteenth century, mycobacterial diseases still constitute a leading cause of morbidity and mortality in countries with limited medical resources. Additionally, mycobacterial diseases can cause overwhelming, disseminated disease in immunocompromised patients. In spite of the efforts of numerous health organizations worldwide, the eradication of mycobacterial diseases has never been achieved, nor is eradication imminent.
Nearly one third of the world's population is infected with Mycobacterium tuberculosis complex, commonly referred to as tuberculosis, with approximately 8 million new cases, and two to three million deaths attributable to tuberculosis yearly. Tuberculosis is the cause of the largest number of human deaths attributable to a single etiologic agent (1). After decades of decline, tuberculosis is now on the rise. In the United States, up to 10 million individuals are believed to be infected. Almost 28,000 new cases were reported in 1990, constituting a 9.4 percent increase over 1989. A sixteen percent increase in tuberculosis cases was observed from 1985 to 1990. Overcrowded living conditions and shared air spaces are especially conducive to the spread of tuberculosis, contributing to the increase in instances that have been observed among prison inmates, and among the homeless in larger U.S. cities.
Approximately half of all patients with “Acquired Immune Deficiency Syndrome” (AIDS) will acquire a mycobacterial infection, with tuberculosis being an especially devastating complication. AIDS patients are at higher risks of developing clinical tuberculosis, and anti-tuberculosis treatment seems to be less effective than in non-AIDS patients. Consequently, the infection often progresses to a fatal disseminated disease. Mycobacteria other than M. tuberculosis are increasingly found in opportunistic infections that plague the AIDS patient. Organisms from the M. avium-intraceliulare complex (MAC), especially serotypes four and eight, account for 68% of the mycobacterial isolates from AIDS patients. Enormous numbers of MAC are found (up to 1010 acid-fast bacilli per gram of tissue), and consequently, the prognosis for the infected AIDS patient is poor.
The World Health Organization (WHO) continues to encourage the battle against tuberculosis, recommending prevention initiatives such as the “Expanded Program on Immunization” (EPI), and therapeutic compliance initiatives such as “Directly Observed Treatment Short-Course” (DOTS). For the eradication of tuberculosis, diagnosis, treatment, and prevention are equally important. Rapid detection of active tuberculosis patients will lead to early treatment by which about 90% cure is expected. Therefore, early diagnosis is critical for the battle against tuberculosis. In addition, therapeutic compliance will ensure not only elimination of infection, but also reduction in the emergence of drug-resistance strains.
Although over 37 species of Mycobacterium have been identified, more than 95% of all human infections are caused by six species of mycobacteria: M. tuberculosis, M. avium intracellulare, M. kansasii, M. fortueitum, M. chelonae, and M. leprae. Cases of human tuberculosis are predominantly caused by mycobacterial species comprising M. tuberculosis, M. bovis, or M. africanum (2). Infection is typically initiated by the inhalation of infectious particles, which are able to reach the terminal pathways in the lungs. Following engulfment by alveolar macrophages, the bacilli are able to replicate freely, with eventual destruction of the phagocytic cells. A cascade effect ensues wherein destruction of the phagocytic cells causes additional macrophages and lymphocytes to migrate to the site of infection, where they too are ultimately eliminated. The disease is further disseminated during the initial stages by the infected macrophages, which travel to local lymph nodes, as well as into the blood stream and other tissues such as the bone marrow, spleen, kidneys, bone and central nervous system. (See Murray et al. Medical Microbiology. The C.V. Mosby Company 219-30 (1990).
There is still no clear understanding of the factors that contribute to the virulence of mycobacteria. Many investigators have implicated lipids of the cell wall and bacterial surface as contributors to colony morphology and virulence. Evidence suggests that C-mycosides, glycopeptidolipids on the surface of certain mycobacterial cells, are important in facilitating survival of the organism within macrophages. Trehalose 6,6′ dimycolate, a cord factor, has been implicated for other mycobacteria.
The interrelationship of colony morphology and virulence is particularly pronounced in M. avium. M. avium bacilli occur in several distinct colony forms. Bacilli which grow as transparent, or rough, colonies on conventional laboratory media are multiplicable within macrophages in tissue culture, are virulent when injected into susceptible mice, and are resistant to antibiotics. Rough or transparent bacilli, which are maintained on laboratory culture media, often spontaneously assume an opaque colony morphology, at which time they are not multiplicable in macrophages, are avirulent in mice, and are highly susceptible to antibiotics. The differences in colony morphology between the transparent, rough and opaque strains of M. avium are almost certainly due to the presence of a glycolipid coating on the surface of transparent and rough organisms, which acts as a protective capsule. This capsule, or coating, is composed primarily of C-mycosides, which apparently shield the virulent M. avium organisms from lysosomal enzymes and antibiotics. By contrast, the non-virulent opaque forms of M. avium have very little C-mycoside on their surface. Both the resistance to antibiotics and the resistance to killing by macrophages have been attributed to the glycolipid barrier on the surface of M. avium. 
The emergence of drug-resistant M. tuberculosis is an extremely disturbing phenomenon. The rate of new tuberculosis cases proven resistant to at least one standard drug increased from 10 percent in the early 1980's to 23 percent in 1991. Compliance with therapeutic regimens, therefore, is also a crucial component in efforts to eliminate tuberculosis and prevent the emergence of drug resistant strains. Equally important in the development of new therapeutic agents that are effective as vaccines, and as treatments, for disease caused by drug resistant strains of mycobacteria. 
Multidrug-resistant tuberculosis (MDR-TB) is a form of tuberculosis that is resistant to two or more of the primary drugs used for the treatment of tuberculosis. Resistance to one of several forms of treatment occurs when bacteria develop the ability to withstand antibiotic attack and relay that ability to their progeny. Since an entire strain of bacteria inherit this capacity to resist the effects of various treatments, resistance can spread from one person to another.
The World Health Organization (WHO) estimates that up to 50 million persons worldwide may be infected with drug resistant strains of tuberculosis. Also, 300,000 new cases of MDR-TB are diagnosed around the world each year and 79 percent of the MDR-TB cases now show resistance to three or more drugs routinely used to treat tuberculosis. According to WHO, nearly 1 billion people will be infected with tuberculosis within the next decade if more effective preventative procedures are not adopted.
In 2003, the CDC reported that 7.7 percent of tuberculosis cases in the U.S. were resistant to INH, a first line drug used to treat tuberculosis. The CDC also reported that 1.3 percent of tuberculosis cases in the U.S. were resistant to both INH and RIF. RIF is the drug most commonly used with INH.
Clearly, the possibility of drug resistant strains of tuberculosis that develop during or before treatment are a major concern to health organizations and health care practitioners. Drugs used in the treatment of tuberculosis include, but are not limited to, ethambutol (EMB), pyrazinamide (PZA), streptomycin (STR), isoniazid (INH), moxifloxacin (MOX), and rifampicin (RIF). The exact course and duration of treatment can be tailored to a specific individual, however several strategies are well known to those skilled in the art.
The standard treatment for tuberculosis caused by drug-sensitive organisms is a six-month regimen consisting of four drugs given for two months, followed by two drugs given for four months. The two most important drugs, given throughout the six-month course of therapy, are INH and RIF. Although the regimen is relatively simple, its administration is quite complicated. Daily ingestion of eight or nine pills is often required during the first phase of therapy; a daunting and confusing prospect. Even severely ill patients are often symptom free within a few weeks, and nearly all appear to be cured within a few months. If the treatment is not continued to completion, however, the patient may experience a relapse, and the relapse rate for patients who do not continue treatment to completion is high. A variety of forms of patient-centered care are used to promote adherence with therapy. The most effective way of ensuring that patients are taking their medication is to use directly observed therapy, which involves having a member of the health care team observe the patient take each dose of each drug. Directly observed therapy can be provided in the clinic, the patient's residence, or any mutually agreed upon site. Nearly all patients who have tuberculosis caused by drug-sensitive organisms, and who complete therapy will be cured, and the risk of relapse is very low (3).
The FDA approved a medication that combines the three main drugs (INH, RIF, and PZA) used to treat tuberculosis into one pill. This reduces the number of pills a patient has to take each day and makes it impossible for the patient to take only one of the three medications, a common path to the development of MDR-TB. Despite this, there is still a need in the art to treat tuberculosis, especially in those cases wherein the tuberculosis strain is drug resistant.
Key to stemming the spread of drug-resistant tuberculosis is the development of rapid and accurate diagnostics to identify MDR-TB and extreme drug resistant tuberculosis (XDR-TB) infection. Gold-standard antibiotic susceptibility tests (AST) require several weeks or months to perform because they measure the growth of this notoriously slow-growing bacteria. Regardless of the time required for conducting AST, these techniques remain important and valuable as they are very accurate because they biologically measure the effect an antimicrobial has on a tuberculosis isolate.
Traditional diagnosis of mycobacterial infection is confirmed by the isolation and identification of the pathogen, although conventional diagnosis is based on sputum smears, chest X-ray examination (CXR), and clinical symptoms. Isolation of mycobacteria on an agar culture plate takes as long as four to eight weeks. Species identification takes a further two weeks. There are several other techniques for detecting mycobacteria such as the polymerase chain reaction (PCR), mycobacterium tuberculosis direct test, or amplified mycobacterium tuberculosis direct test (MID), and detection assays that utilize radioactive labels. Most of these tests are often cumbersome, require a high level of technical expertise and require long periods of time before useful results can be obtained.
One diagnostic test that is widely used for detecting infections caused by M. tuberculosis is the tuberculin skin test. Although numerous versions of the skin test are available, typically one of two preparations of tuberculin antigens are used: old tuberculin (OT), or purified protein derivative (PPD). The antigen preparation is either injected into the skin intradermally, or is topically applied and is then invasively transported into the skin with the use of a multiprong inoculator (Tine test). Several problems exist with the skin test diagnosis method. For example, the Tine test is not generally recommended because the amount of antigen injected into the intradermal layer cannot be accurately controlled (4).
Although the tuberculin skin tests are widely used, they typically require two to three days to generate results, and many times, the results are inaccurate since false positives are sometimes seen in subjects who have been exposed to mycobacteria, but are healthy. In addition, instances of misdiagnosis are frequent since a positive result is observed not only in active tuberculosis patients, but also in persons vaccinated with Bacille Calmette-Guerin (BCG), and those who had been infected with mycobacteria, but have not developed the disease. It is hard, therefore, to distinguish active tuberculosis patients from the others, such as household tuberculosis contacts, by the tuberculin skin test. Additionally, the tuberculin test often produces a cross-reaction in those individuals who were infected with mycobacteria other than M. tuberculosis (MOTT). Therefore, diagnosis using the skin tests currently available is frequently subject to error and inaccuracies.
Traditional methods to identify drug resistant strains of M. tuberculosis (Mtb) involve culturing Mtb isolated from clinical specimens in either liquid culture or on solid supports such as LJ slants or agar plates supplemented with the appropriate nutrient media for growth of mycobacteria. After the culture reaches a sufficient population density allowing visual identification of bacterial growth either by an increase in turbidity of a liquid culture or by colony formation on LJ slants or agar plates, the isolate is sub-cultured into two or more individual vessels either containing a different antibiotic or none at all. The effect of the antibiotic on the Mtb isolate is determined by comparing the growth of the antibiotic-containing subculture to one in which no drug was added. If the Mtb isolate is susceptible to the antibiotic, it will not grow sufficiently compared to the control. However, if the strain is resistant to the anti-tuberculosis drug, then it will continue to grow as it has the ability to circumvent the antimicrobial properties of the antibiotic. Second-generation versions of this biological, growth-based assay speed the time to detection of both microbial identification as well as resistance testing by using radiometric (e.g. Becton Dickinson's BACTEC™) or colorimetric (e.g. Becton Dickinson's MGIT™ and Biomerieux's BACT ALERT®) devices to measure or CO2 produced by growing Mtb, rather than waiting for the bacterial population to reach a density sufficient to be seen by the naked eye.
More recent approaches to speed the biological detection of drug resistance in Mtb have focused on lysing mycobacteriophage to probe the effect an anti-microbial has on an Mtb isolate. Mycobacteriophage are viruses that infect Mycobacteria and hijack cellular biosynthetic machines to replicate and spread. Broadly speaking, viruses are obligate intracellular parasites: they rely on the biosynthetic machinery of the host cell to manufacture progeny in order to reproduce and spread. The extent to which an infecting virus is able to direct the synthesis and production of new viral components is largely dictated by the metabolic capacity (i.e. the viability status) of the host cell. In the extreme version, a living cell, which is metabolically active, can be infected by a virus and be co-opted for the production of new virus while a dead, metabolically inactive cell, cannot. Therefore, the extent to which a virus is able to infect and replicate in a host cell indicates the metabolic capacity of that cell. Because antibiotics ultimately affect the metabolic capacity of a susceptible bacterial cell, and phage hijack the biosynthetic machines of the host, antibiotics also have an effect on the replication and spread of bacteriophage. Furthermore, phage are able to find and infect small numbers of bacteria and because they replicate and spread so much faster than the host cell, especially in the case of Mtb, they can be used to dramatically speed the detection and antibiotic susceptibility test (AST) of Mtb.
Biotec, Inc.'s (Suffolk, United Kingdom) product FASTPLAQUE-RESPONSE™ measures the ability of the mycobacteriophage D29 to replicate inside Mtb after exposure to antibiotics. Mtb isolated from a clinical specimen are split into two vessels: one contains the anti-microbial rifampicin (RIF) while the other contains no antibiotic and serves as a positive control. After RIF is given sufficient time to exert its effects on Mtb, D29 is added to both tubes and given sufficient time to inject its DNA into Mtb and hijack the cell to make progeny virus. Prior to lysis of Mtb by phage-encoded lysis functions, extracellular phage that did not infect Mtb are killed by addition of a chemical virucide, which cannot penetrate inside Mtb and therefore kills all extra-cellular phage. The virucide and antibiotic are then removed and a fast-growing mycobacteria, M. smegmatis (Msmeg), is added to the phage-infected Mtb. The mixture is then plated onto agar dishes. Because Msmeg replicates quickly, a bacterial lawn is formed on the agar plates after overnight incubation at 37° C. Furthermore, Msmeg is efficiently infected by D29, which forms clear and visible plaques on Msmeg bacterial lawns. Each plaque represents an Mtb cell that was initially infected by D29 and produced progeny phage. This assay quantitatively measures D29 replication in small numbers of Mtb. Furthermore, because phage replication is wholly dependent upon the metabolic capacity of the host cell, quantitative measurements of D29 replication in Mtb exposed to an anti-microbial compared to an untreated control accurately measures the extent to which that antibiotic can disrupt Mtb metabolism, and ultimately bacterial growth. Low viral replication reflects antibiotic-mediated inhibition of cellular metabolism, whereas high viral replication biologically demonstrates Mtb drug resistance. Finally, this phage-based drug resistance detection assay is a biological test as it directly measures the biological effect a drug has on tuberculosis. While previous biological tests were growth based and took several weeks to identify a difference in bacterial growth between drug treated and untreated samples, FASTPLAQUE-RESPONSE™ is a rapid biological test that does not measure cell growth. Instead, it uses the mycobacteriophage D29 to measure an antimicrobial's effect on host cell metabolism. Although an accurate and rapid test, FASTPLAQUE-RESPONSE™ is too complicated and unwieldy for use in resource-poor settings because the analysis of viral growth by plaque formation on agar plates must be performed in a laboratory by a highly-trained technician.
Another phage-based system for the detection of Mtb drug resistance measures the enzymatic activity of a single phage-encoded polypeptide rather than the ability of the entire mycobacteriophage to replicate and spread as is done by FASTPLAQUE RESPONSE™. This system was originally developed by researchers at Albert Einstein College of Medicine and is known as the Luciferase Reporter Assay (LRA). The LRA utilizes a recombinant version of the mycobacteriophage TM4 that has been engineered to highly express the luciferase gene from the firefly Photinius pyralis. Luciferase is a single subunit enzyme that, upon cleavage of its substrate luciferin in the presence of ATP and molecular oxygen, releases a photon of light. The presence of luciferase can thus be measured by detecting its light production. During Luciferase Reporter Phage (LRP) infection of untreated Mtb, the luciferase polypeptide accumulates and its enzymatic activity can be detected by measuring photon production after adding luciferin, which readily enters Mtb cells. Incubation of drug susceptible Mtb with appropriate anti-tuberculosis antibiotics either kills the cell outright or leads to a decrease in the metabolic capacity of the cell. Because adenosine triphosphate (ATP) is the essential source of potential energy in the bacteria and luciferase activity requires ATP for not only enzyme activity but also for the luciferase enzyme's synthesis, luciferase activity is an indicator of a bacteria's metabolic capacity and hence the effect a given anti-microbial has on bacterial viability. During LRP infection of drug susceptible Mtb treated with an anti-tuberculosis antibiotic, luciferase enzyme synthesis and subsequent light production is dramatically reduced compared to an LRP infected control to which no anti-tuberculosis antibiotic was added. This differential in luciferase activity demonstrates an antimicrobial's effectiveness against Mtb. However, if light production in drug-treated and LRP infected Mtb is similar to an untreated control, the Mtb isolate is identified as drug resistant. The LRA has been evaluated in clinical trials testing Mtb resistance to first line anti-microbials and shown to have greater than 90% sensitivity and 100% specificity. Although an excellent tool to speed detection of drug resistant Mtb, only very sophisticated luminometers can detect luciferase light production from the small numbers of bacteria present in a clinical specimen. The LRA is thus not amenable to use in resource-poor settings that do not have the capacity to purchase and operate a high quality luminometer.
Recent efforts to rapidly identify drug resistance directly from clinical specimens employ nucleic acid amplification (NAA) to detect specific Mtb genomic loci that confer resistance to commonly used anti-tuberculosis drugs. NAA is a process by which a nucleic acid sequence is selectively replicated using enzymatic methods to increase the number of identical nucleic acid sequence molecules and thereby increase the sensitivity of the assay. Many but not all examples of nucleic acid testing (NAT) use NAA. NAT is the detection of nucleic acids using methods such as molecular binding, hyrbidization, fluorescence, chemiluminescence, and radioactivity to specifically or non-specifically detect nucleic acids. One example of a NAT that employs NAA is Hain Lifescience' s (Nehren, Gennany) GENOTYPE® MtbDR, which uses a line probe assay to detect specific drug resistance alleles amplified from clinical sample-derived Mtb DNA. GENOTYPE® MtbDR is proving very complicated and expensive as there are over 15 commonly observed known mutations that confer resistance to RIF and INH.
Another molecular diagnostic technology in development by Cepheid (Sunnyvale, Calif.) is intended only for diagnosis of RIF resistance in Mtb. Cepheid's market advantage is mostly due to the GENEXPERT® system, which fully automates sample processing and NAA. However, detection of the individual resistance loci in the amplification reaction requires fluorescent probes that are expensive to synthesize and require sophisticated detection hardware. Because of this limitation, the Cepheid product is limited to detecting the five major mutations involved in RIF resistance. For the Cepheid system, simultaneous detection of both RIF and INH resistance loci would be too unwieldy and expensive.
All currently available molecular diagnostic technologies fail to satisfy today's need for effective diagnostics as they are incapable of detecting Mtb strains that are RIF or INH resistant but harbor uncharacterized mutations. They also fail to identify isolates resistant to other first-line antibiotics, much less XDR-TB strains, because the full gamut of clinically relevant mutations conferring resistance to all anti-Tb drugs is not known. A rapid molecular diagnostic test able to identify all drug-resistant Mtb strains, including emergent XDR-TB, will be an important and necessary tool for the effective treatment and control of drug-resistant tuberculosis. The development of such rapid molecular testing technology would also be relevant and important for other diseases including, but not limited to, cholera, cryptosporidiosis, leishmaniasis, meningitis, and pneumonia. Additionally, the development of accurate molecular testing enabling the detection of microbes would also be useful for the detection of contaminants in pollutants ranging in sample type from drinking water to laboratory reagents.
What are needed are effective diagnostic and therapeutic tools to address the ever persisting and ever evolving challenges posed by infectious disease, in particular mycobacterial disease. In addition, as the use of antibiotics becomes increasingly widespread, and in some cases where the use of antibiotics is not in compliance with prescribed and recommended regimens, we find ourselves challenged with novel strains of infectious agents and microbes that no longer respond to standard therapies. What is needed therefore, are effective tools for identifying infectious agents, wherein such tools are also preferably capable of determining drug susceptibility. Importantly, what is needed are diagnostic tools that are easy to use, that require minimal testing time, and that are inexpensive so that they are readily available for use in parts of the world where the disease is prevalent, and where resources are limited.
What are also needed are efficient, simple and accurate molecular testing technologies that enable the detection of infectious agents, that provide information concerning the viability of the infectious agent and that determine drug susceptibility. Use of such technologies would not be limited to infectious disease alone; their utility could be extended to detection and evaluation of microbes and pollutants in a variety of samples ranging from biological to industrial.